Bacteriophages or plasmids are required for the synthesis of toxins in several pathogenic bacteria. The long-range goal of our studies is to determine the genetic and biochemical mechanisms that regulate toxinogenesis in such systems. The production of diphtherial toxin by Corynebacterium diphtheriae will be used as a model for phage-directed toxin synthesis. The regulation of synthesis of enterotoxin in Vibrio cholerae will be analyzed and compared with the diphtherial system, and the possible role of plasmids in determining production of cholera enterotoxin will be investigated. Our goals in the analysis of the diphtherial system are to isolate and characterize mutants of corynebacteriophages B and of C. diphtheriae C7 that alter toxinogenesis, establish whether such mutants have phenotypes that are consistent with current theory for the regulation of toxinogenesis in C. diphtheriae, to analyze the formal genetic mutations in phage B that affect toxinogenesis, and to determine whether specific systems regulating toxinogenesis operate by controlling transcription of the structural gene tox or by modifying translation of toxin-specific messenger RNA. Our objectives in analyzing toxinogenesis in V. cholerae are to identify and characterize mutations in the structural and regulatory genes controlling enterotoxin production, to analyze structure-activity relationships in wild type and mutant enterotoxin proteins, and to study representative mutants altered in toxinogenesis by recombinational analysis utilizing the conjugal mating system controlled by the vibrio sex factor. The methods developed originally to isolate and characterize tox mutants of V. cholerae will be applied for the selection of strains that have optimal characteristics for use as possible Vibrio cholerae living attenuated vaccines.